A detailed understanding of human health and disease requires methods to probe cellular behaviors as they occur within intact organ structures and living subjects. In recent years, technologies have emerged from the imaging community that enable diverse biological features to be visualized and tracked in real time. While powerful, these approaches have been largely confined to monitoring cellular behaviors on a microscopic level. Visualizing cellular functions across larger spatial scales-including those involved in cancer progression and migration-requires new imaging tools. The long-term goal of our work is to develop general strategies for macroscopic, multi-cell tracking in living organisms. The objective of this application is to engineer novel bioluminescent tools for multi-cellular imaging in vivo. Bioluminescence imaging is a powerful technique for visualizing small numbers of cells in rodent models. This technology employs enzymes (luciferases) that produce light upon incubation with small molecule substrates (luciferins). Several luciferase-luciferin pairs exist in nature, and many have been adapted for tracking cells in whole animals. Unfortunately, the optimal luciferases for in vivo imaging utilize the same substrate, and therefore cannot be used to distinguish multiple cell types in a single subject. Our central hypothesis is that the substrate-binding interface of firefly luciferase can be re-engineered to generate a panel of mutant enzymes that accept chemically distinct luciferins. When the mutants and analogs are mixed together, robust light emission will be produced when complementary enzyme-substrate partners interact. Guided by strong preliminary data, our work will encompass the following specific aims: 1) Synthesize and identify light-emitting luciferins; 2) Generate complementary luciferases and screen for orthogonal pairs; and 3) Image tumor heterogeneity with orthogonal probes. Under the first aim, we will utilize divergent chemistries developed in our lab to access light-emitting small molecules. In the second aim, we will employ a combination of mutagenesis and screening assays to identify luciferase enzymes that catalyze light emission with the synthesized molecules. In the third aim, the enzyme-substrate pairs will be utilized to address the roles of distinct cellular subsets in heterogeneous tumor models. Our approach is highly innovative, as it combines a unique blend of chemical and biological techniques to fill a long-standing void in imaging capabilities. The proposed research is significant, as the bioluminescent tools will enable the direct interrogation of cell networks not currently possible with existing toolsets. Such studies will provide some of the first macroscopic images of tumor heterogeneity and may fundamentally change existing views on cancer progression and therapeutic approaches. Additionally, similar to other imaging technologies, the bioluminescent probes will likely inspire new discoveries in a broad spectrum of fields.